Continuous antidromic electrophoresis



May 12, 1964 s. NATELSON 3,133,009

CONTINUOUS ANTIDROMIC ELECTROPHORESIS Filed Nov. 13, 1959 2 SheetsSheet 2 Abs.

Th L I1 V i 2 o-w- 5 10 I5 20 Cm- Abs- INVENTOR. I Snnuen. mason .4 77-02 NEY United States Patent Ofifice 3,133,009 Patented May 12, 1964 3,133,0tl9 CONTINUOUS ANTiDRfih EC ELECTROPHORESE Samuel Natclson, Brooklyn, N.Y., assignor to Scientific Industries, Inc., Springfield, Mesa, a corporation of Delaware Filed Nov. 13, 1959, Ser. No. 852,811 6 Claims. (ill. 204-299) The present invention relates to the separation of chemical substances and more particularly to the separation of substances with similar properties such as homologues, isomers and isotopes.

It is well known that electrophoresis teaches the separation of chemical substances by passing an electric current through a butler supported on a neutral medium, e.g., a paper tape wetted with bufier on which the substances have been placed. In general, the positive ions in the substances move towards the negative electrical terminal and the negative ions towards the positive terminal thereby separating the ingredients. However, when the substances to be separated have similar chemical properties with mobilities only slightly dillerent from each other, e.g., certain amino acids, the separation of such substances by Zone or paper electrophoresis presents a serious problem to the analyst. Attempts have been made to use twodimensional electrophoresis using both acid and alkaline buffers. A combination of paper chromatography with electrophoresis has also been tried. Still others have used high voltage electrophoresis between two large glass plates immersed in a cooling organic solvent. But, in no case has a clear separation of similar substances differing only slightly in mobility been achievable by means of electrophoresis alone.

One of the major problems of long range electrophoresis is the phenomenon of electrophoretic equilibrium or mo bility equilibrium. If electrophoresis is performed in a freely suspended stationary strip of paper, heat is generated and evaporation takes place. The buffer then flows from both electrodes towards the center of the strip or tape. Thus, if one stripes a marker (e.g., malachite green) near the negative electrode towards which it should migrate in an acid buffer, it will actually move backward toward the center of the strip, being pushed back by the buffer flow. On the other hand, if this substance is placed near the positive electrode, it will move much more rapidly than if placed at the center. If the current flows long enough, the two samples of malachite green will converge to form one stain and assume a position of equilibrium where its rate of migration is just balanced by the buffer flow. This has been demonstrated for amino acids (Durrum, E. L., J. Am. Chem. Soc., vol. 73,-page 4875, 1951). In attempting to separate amino acids therefore, once this time has been reached, there is no advantage obtained from further duration of the period of electro phoresis. Of greater difiiculty is the fact that the pattern becomes compressed. This is so because if nearer the negative electrode, for amino acids in acid buffer, the faster amino acids are slowed down more than those hehind. 'If nearer the positive electrode, the slower amino acids are pushed ahead by the flowing butter. Further, the pattern obtained is not reproducible in that the rate of migration will depend upon where the sample is first striped, for periods of time less than that required to reach electrophoretic equilibrium.

Although many attempts were made to overcome the foregoing difficulties and other disadvantages, none, as far as I am aware was entirely successful when carried into practice for any practice application.

It is an object of the present invention to provide an apparatus which will readily separate chemical substances, particularly substances with similar chemical properties and close electrical characteristics and with slightly diilerent from each other.

Another object of the present invention is to provide a method for the separation of chemical substances, particularly substances with similar chemical properties and with mobilities only slightly different from each other.

The invention also contemplates providing a simple and convenient way of separating substances.

It is also the purpose of the invention to provide an apparatus for the aforesaid purposes which is inexpensive to manufacture, of simple construction, and which can be operated by untrained or unskilled personnel.

With the foregoing and other objects in view, the invention resides in the novel arrangement and combination of parts, in the details of construction, and in the process steps hereinafter described and claimed, it being understood that changes in the precise embodiments of the invention herein disclosed may be made within the scope of what is claimed without departing from the spirit of the invention.

In its broader aspects, the invention contemplates an instrument which continuously moves an endless, electrically conductive supporting medium in a direction opposite to the migration of the ions, the ions migrating indefinitely until complete separation has been achieved.

The invention will appear more clearly from the following detailed description when taken in connection with the accompanying rawings, showing by way of example, a preferred embodiment of the inventive idea.

FIG. 1 is a longitudinal view of one type of apparatus contemplated herein;

FIG. 2 illustrates a longitudinal view of another type of apparatus contemplated herein;

FIG. 3 illustrates the results obtained on a densitometer when recording a tape having thereon certain amino acids with closely related mobilities which have been separated by the apparatus depicted in FIG. 2 and stained; and

FIG. 4 illustrates the results obtained on the same densitometer when recording a tape having thereon certain other amino acids with closely related mobilities separated by the apparatus depicted in FIG. 2.

mobilities only Generally speaking, the present invention contemplates an instrument for the continuous separation of substances with similar but not identical electrophoretic mobilities by placing the substances to be separated on an electrically conductive supporting tape. An electrical direct current is passed through an elongated portion of the tapetowards the central area of which elongated portion said substances to be separated have been placed, and the tape moved continuously by mechanical means in a direction opposite to the migration of the ions at a rate of speed somewhat of the order of the speed of migration of the ions of the substances being separated for an extended hourly period of time. This tape may have the edges impregnated with plastic, e. g., polyethylene, and have sprocket holes cut therein for accurate guidance. The impregnation with plastic strengthens a fragile tape such as paper. Or, rollers may be used.

According to one embodiment of the invention, there is provided an elongated box 10, preferably made of noncondutcive electrical material, e.g., wood or plastic. A fiat piece of plastic or glass not shown in the drawing acts as a cover for the box. At one end of the box is a feeding reel ill. This reel holds the supporting medium which in the case of this embodiment is Whatman 3 paper 1 /2 inches wide; although, of course a plastic film supporting a gel would do equally well. A cord made of nylon, silk, cotton, or other fiber thread may also be used for the same purpose. To better illustrate the invention, supports for feeding reel 11 have been omitted from the drawing.

The supporting medium 12 which is usually wound around reel 11 beforehand is fed through slits 13, through guide rollers 14 and 15, made of non-conductive electrical material, e.g., polyethylene. To apply tension to the supporting medium, the rollers are held together with a spring 16. From the guide rollers 14 and 15, the tape passes to the first electrical contact roller 17. In the embodiment shown in FIG. 1, this roller was made of polyethylene around which filter paper had been wrapped and sewn in place" to act as a wick. Sintered glass rollers may be used for the same purpose. Roller 1'7 is so designed as to continuously supply buffer solution to the supporting medium, i.e., tape 12. The buffer solution 18 rests in a container 18a at each end of box 10. Electric power is supplied by power supplying means shown in block diagram in the drawing. The power supply for this embodiment is adequate if it can supply filtered DC. voltage without ripple of the order of 90 to 500 volts. Associated with the power supply are the customary positive and negative terminals 21 which are adapted and designed to engage corresponding terminals in the frame. Attention is directed to the fact that the terminals or sockets 21a and 21b in the frame are not identified as being positive or negative. This is because of the fact that the movement of the tape is unidirectional. And, since the direction of movement of the tape must be counter to the direction of migration of the ions of the substances being separated, the connection of the power supply terminals to the frame sockets 21a and 21b depends on the substances being separated. The current from sockets 21a and 21b is fed to electrodes 2% located in buffer solution 18. These electrodes advantageously may be made from platinum wires wound on a Lucite or polyethylene bar. Since the action of the electricity from the electrodes 2!) in the buffer solution 18 produces substances, it is essential that these substances which are impurities be prevented from getting on to the tape 12 so as not to interfere with the separation of the substances on the tape. This is accomplished by means of a bafiie plate 19 which isolates the electrode. This bafile plate is supported in the vicinity of r the electrode in the buifer solution a few millimeters from the bottom of the soltuion and thus allows electrical continuity. Thus, the electricity goes from the power supply to the terminal 21a; to the electrode 20 supplied by terminal 21a; along the bottom of the bufier 18; up wick roller 17, along tape 12 as far as the corresponding wick roller 17a which is identical to the first described wick roller 17; down wick roller 17a to buffer 13 on the far side of the box, the buffer solution 18 on the near and far side being separated by a partition 18a; to the electrode 20 on the far side, and back to the power supply. On the far side of the box, tape 12 passes through slits 13a to guide rollers 22 and 23 which are constructed similar to rollers 14 and 15. Guide rollers 22 and 23 are likewise retained by a spring 24 to apply tension to the tape which is wound on take up reel 26 by a slow moving clock motor 29. Preferably, the motor 29 acts on guide roller 22 which is a constant speed roller pinned to gear 25 which is activated by gear 28 whose rate of rotation is in turn controlled by the constant speed motor, i.e., clock motor 29. A gear train arrangement is provided in connection with motor 29 which is adjustable, i.e., the gears may be shifted to change the speed. This type of motor is well known. a spring pulley 27 in engagement with roller 22. If instead of a liquid buffer, a plastic film tape supporting a gel is used, the apparatus is the same except that the gel layer is placed on the bottom of the tape to make contact.

This embodiment uses low voltages and currents so that the heat generated will not evaporate the liquid buffer medium, e.g., water and thus break the circuit.

In this embodiment, the substances to be separated are placed towards the center of the tape and the phenomenon of electrophoretic equilibrium is partially overcome by moving the paper at a rate designed to keep the amino acids in the same position relative to the electrodes and as far from both electrodes as possible, i.e., near the center.

rollers 34a and 34b are on both sides The take up reel 26 is turned by tively longer run than the second embodiment herein described. If a thickening agent such as methoxy cellulose or starch is added to the aqueous buffer, evaporation is retarded and somewhat higher voltages may be used.

A better arrangement is that shown in the second embodiment which utilizes a cooling medium so that higher currents and voltages may be used, achieving the desired separation in a shorter .time. As is apparent from the drawing, in the instrument depicted in FIGURE 2 cooling means are provided.

Thus, there is provided a feed reel 31 containing the tape or supporting medium 33, i.e., a paper or gel, and fed into a box 35, advantageously a Teflon coated aluminum box or frame. Towards the opposing ends of the box are contact wick rollers 32a and 3215 whose function is identical to contact wick rollers 17 and 17a hereinbefore described in conjunction with the first embodiment. On both sides of the center of the instrument are idler rollers 34a and 34b. In the embodiment shown in the drawing, these are Teflon 1" diameter rollers. Under each contact wick roller 32a and 32b is some buffer 44 solution, e.g., an aqueous solution, 1.5 normal with respect to acetic acid and 1 normal with respect to formic acid. As hereinbefore described, the electric current goes from one of the terminals, e.g., 45a to the electrode 47, under the bathe plate 46 designed to keep impurities from reaching the tape 33, up roller 32a, along the tape to the other contact wick roller 32b, into the butter 44, under baffle 46, to electrode 47 and out through terminal 45b. In FIG. 2 depicting this embodiment, the power supply has been omitted. On the far end of the instrument are pressure rollers 36 and 36a advantageously Tygon coated. Pressure on the tape is maintained by spring 37 thus keeping the tape level and without slack. The tape is wound on take-up reel 38. The tape may be wound on this reel by an arrangement shown in the drawing, namely, the motor 43 which is a very slow clock type arrangement moves gear 42 at a speed of 1-l4 inches per hour, the speed of course being adjustable depending on the material being separated. Gear 42 in turn engages corresponding gear or pinion .40 which is connected to take-up reel 38 by means of spring pulley 39. An important feature of this embodiment is the cooling chamber 51 made of glass or polyethylene. This chamber is an elongated chamber disposed at right angles to the principal axis of the frame and in the center of the frame. The guide of chamber 51 and guide the tape in and out thereof. At the end of the chamber is another roller 50 to reverse the direction of travel of the tape. This roller 50 is maintained by Teflon coated metal rods 49. A cooling coil 48 runs through the chamber. Tap water or a refrigerated liquid is moved through cooling coil 48, not in electrical contact with the system. When a heat transfer, inert and insulating fluid 52, e.g., carbon tetrachloride, Freon, or octane is in the chamber, the tape 33 and substances or buifer'solution thereon will be cooled. By the use of this cooling chamber, much higher currents and voltages (of the order of 500 to 3000 volts) can be used than in the first embodiment described. The use of high currents and voltages materially reduces the time for separating the substances. Attempts to use cooling by circulating cooled substances to be separated and must be so that the direction of travel of the ions is opposite to the direction of travel of the tape. Thus, by moving the tape at the approximate speed of migration of the ions in-the substances being separated, the ions remain substantially in the same placet: Where many substances are present the pattern develops with time, some substances moving to one side ofroller 50, some remaining in the center of roller 50 and some to the other side of roller 50, the separation spreading with time.

In connection with the second embodiment, it is preferable to use a paper tape immersed in carbon tetrachloride as the cooling medium. There is a much reduced buffer flow due to evaporation up to the surface of the carbon tetrachloride and it stops there. Thus, for the portion of the strip of tape immersed in the carbon tetrachloiide, the rate of migration is almost linear with time, and the longer one runs the current, the wider is the separation, provided the specimen remains submerged in the carbon tetrachloride. Runs up to 48 hours have been made with the instrument requiring minimal attention. The instrument may be safely allowed to run overnight unattended. In practice, a strip of tape has been immersed in 45 cm. of carbon tetrachloride. Thus, the pattern may develop over 90 cm. of paper. Further, it is preferable to have some additional cm. of paper on each side in air available before the contact roller is reached. Thus, the total field is 115 cm. over which the substances, e.g., amino acids may spread. During a 24 hour run, 192 cms. of paper is pulled through the field. In order to duplicate such a set up with a stationary run, one would need an instrument of 307 cm. length between electrodes. It would also be necessary to have siliconed glass pressure plates of this length and a voltage in excess of 5000 volts as compared with the 1800 to 2000 advantageously used in connection with the instrument depicted in FIG. 2. The overall length of this instrument from electrode to electrode is only 18 cm. as compared with 307 cm. required for a stationary instrument. If one were to compare a 48 hour run on this instrument with a stationary set up one would have to double the voltage (in excess of 10,000 volts) and more than double the length (499 cm. total).

For the purpose of giving those skilled in the art a better understanding of the invention, the following illustrative example is given:

Example The instrument described in connection with FIG. 2 was used for the separation of amino acids. All operations involving the handling of the paper were done using rubber gloves to avoid contamination. The instrument used was in a completely enclosed case. The drawings do not show the case merely to better depict the instrument.

Four meters of Whatman #3 paper 1 /2 inches Wide were wound on a removable reel. The reel was dipped in the butter and the excess solution shaken oil. In this case,

the buffer comprised 80 ml. glacial acetic acid and 46 ml. of formic acid diluted to make one liter with Water. The reel was then replaced and the cooling chamber containing the carbon tetrachloride was removed. Regarding the carbon tetrachloride, this was made iron free by allowing commercial carbon tetrachloride to stand over anhydrous sodium sulfate overnight, filtered through glass wool and distilled in an all glass apparatus. Unless the iron is removed from the carbon tetrachloride, the iron will aifect the results obtained as hereinafter explained.

The moist paper was then drawn over contact roller 32a, idler guide rollers 34a, 50, and 34b, over second contact roller 32!) and under pressure roller 36 which was lifted and the paper pulled through. The paper was wound on take-up reel 38 and reel 31, the feed reel was turned back until the paper was taut on the rollers. Excess moisture was wiped off from the paper with gauze, and a mixture of amino acids was applied with a pipet at the bottom of roller 50.

The carbon tetrachloride containing tank or chamber 51 was then replaced and a voltage of 17 v./cm. (approx. 2000 volts) was applied. The motor was started and set to run at 8 cm. per hour for the so called acidic and neutral amino acids and at 15 cm. per. hour for the basic amino acids. The actual rate depends on the buffer used. Tap Water was allowed to flow through the coil and into a drain at a rate suitable for maintaining the carbon tetrachloride at room temperature (approx. 22-25 C.). Satisfactory runs for the separation of the 15 most common acidic and neutral amino acids took from 18 to 24 hours. At the end of the run, the current was disconnected, the carbon tetrachloride tank or chamber removed, and the paper Was slit at the contact rollers. The paper was dried in an oven maintained at 75 C. on cotton gauze or suspended from Teflon supports. The dried paper was then dipped into a color developing mixture and again allowed to dry and heat at 75 C. until the colors were developed some 10 to 15 minutes after drying.

As a color staining solution 2 grams of ninhydrin and 300 mg. of hydrindantin were dissolved in 500 ml. of absolute ethyl alcohol. This reagent is stable in the refrigerator indefinitely. Just before use, 10 ml. of glacial acetic acid and 8 ml. of collidine were added to 50 ml. of this solution.

A curve was prepared using a densitometer with 1 mm. light slit with suitable light filters, by passing the paper through and plotting absorbance vs. distance. The results are shown in FIGS. 3 and 4 which are typical of such curves. Concentration is estimated by the area under the curves as compared with a standard for a particular amino acid.

While the instrument of FIG. 1 may be made of Lucite, this is not so for the instrument of FIG. 2, because of the effect of solvents on Lucite. Teflon is preferred, but polyethylene is satisfactory and much less expensive. Thus, the rollers, frame and containers,'including the chamber or jar for holding the carbon tetrachloride may all be made of polyethylene. No iron may be present in the system since it will dissolve and serve to increase the con ductivity thus resulting in overheating. Further, iron will change the color of the stains and cause fading. For these reasons, the axles on which the rollers turn are made of Teflon and the carbon tetrachloride needs to be redistilled. Even analytical grade of carbon tetrachloride often contains some iron.

Where a mixture of all the common amino acids are present two runs may be performed simultaneously with two instruments but with one power supply. For the basic amino acids (arginine, lysine, histidine) the take-up reel is run at approximately 15 cm. per hour, the exact rate varying with the pH and the concentration of the buffer. The neutral and acidic amino acids pile up at the positive electrode and the three amino acids are isolated in the vicinity of the center roller 50 of FIG. 2. In the second instrument the take up reel is run at 8 cm. per

hour. In this case the basic amino acids are removed from the field, piling up at the negative electrode.

With a single instrument, two sequential runs may be made. At 8 cm. per hour for the take up reel, the neutral and acidic amino acids are kept in the field and separated, the paper is cut at the carbon tetrachloride interface, on the negative side, for development of color. A fresh strip of paper is sewn to the remaining paper near the negative contact. The paper is pulled through to place the basic amino acids around the center roller 50 and electrophoresis is continued for their separation.

The heat transfer medium contained in the jar 51 of FIG. 2 is preferably one which is heavier than Water and not flammable. Further, it should have some volatility otherwise it will slowly creep up the paper and eventually foul the electrodes. It should not react with or dissolve the material being analyzed, under the conditions of the experiment and should not affect the staining process. Carbon tetrachloride, free of iron is suitable for the sep- CChE-CCIF were tested. The aliphatic hydrocarbons have a tendency to creep on the paper and are a fire hazard. The fluorinated hydrocarbons have no advantage over carbon tetrachloride for this particular purpose.

The instrument of the example was operated in a cold room at various temperatures down to 4 C., no advantage was found in the degree of separation. A disadvantage, was the collection of moisture at the surface of the carbon tetrachloride with resulting partial shorting of the system. A further disadvantage was turbulance produced in the pattern probably due to the steep temperature gradient between the outer surface of the jar or chamber and the paper. This caused more rapid circulation of the carbon tetrachloride with resultant disturbance of the patterns.

The portion of an electropherogram of ninhydrin stained paper strip shown in FIG. 3 illustrates the separation of threonine, leucine, isoleucine and valine by the instrument of the example, i.e., of FIG. 2, the buffer being 80 ml. glacial acetic acid and 46 ml. formic acid to one liter with water. The plot is that of absorbancies vs. distance on paper in centimeters. Alanine and glycine are to the right of the diagram shown, widely separated. The factors of importance are: time, 20 hours; 17 v./cm.; take up speed 8 cm./hour. FIG. 4 should be integrated with FIG. 3 and shows a portion of an electropherogram of ninhydrin stained paper strip illustrating the separation of tyrosine, aspartic acid, cystine, phenylalanine, glutamic acid, methionine, proline and threonine.

The scan of the electropherogram separating leucine, isoleucine and valine as shown in FIG. 3 illustrates the efficiency of the instrument in achieving separations which have not been possible before by electrophoresis. On the original strip, three separable distinct spots are observed. FIG. 4 complements FIG. 3 in that the slower neutral and acidic amino acids are clearly separated. Each amino acid is distinctly separated as seen on the original paper. White light was used in the scanner. For higher peaks,

, tyrosine, cystine, phenylalanine and proline which produce colors of lower absorbancies with white light need to be read with different filters. The amino acids of FIG. 3 are readily separated from those of FIG. 4 on the same strip, the former being much more mobile. Threonine is included in FIGURE 3 to integrate this pattern with that of FIGURE 4. Similarly, tryptophane, glycine and alanine run much more rapidly than valine and are readily separated on the same strip. By proper choice of bufier, serine can be made to run either between threonine and leucine, or isoleucine and valine, on the same strip. The relative mobilities of the amino acids is thus markedly affected by change in pH and concentration of bufiers.

It is to be observed therefore that the present invention provides for an apparatus or instrument for the separation of substances whose electrical ions have slightly different rates of electrophoresis mobility or migration, e.g.,

certain amino acids, comprising in combination; an elongated frame adapted to define the limits of an electrically conductive absorbent carrier medium or tape 13 and 33; feed means 11 and 31 at one end of said frame to contain and feed out a continuous even supply of said carrier medium; guide means along said frame to guide said carrier medium along the length thereof; electrical terminal 21a, 21b or 45a, 45b, including circuits associated V with said frame, adapted to provide positive and negative electricity to opposing ends of said frame, either terminal being adaptable to be either positive or negative; 21 contact 17, 17a, 32a, 32b, engaging said carrier medium at each of said opposing ends, associated with each of said terminal circuits; take-up means 26 and 38 at the other end of said frame adapted to take up and retain said carrier medium; and slow moving motor means 29 and 43 for continuously moving said carrier medium at a uniform slow speed somewhere of the order of the speed of the electropheresis migration of the chemical substances to be separated.- Advantageously, there can also be provided a cooling means intermediate said contacts in the path of travel of said carrier medium for cooling said carrier medium, e.g., a cooling chamber 51 having electrical- 0001- ing elements 48 therein. By placing an absorbable mixture of substances whose ions have similar but not identical electrophoresis migratory mobilities, e.g., amino acids, on said absorbent carrier medium towards the center of said frame, passing a direct'electrical current through said terminals and circuits, and moving said carrier medium in a direction opposite to the direction of movement of the ions of said substances at a speed of the order of the rate of electrophoresis migration of said ions for a sufficient period of time, separation of said substances is achieved.

The cooling chamber may take on different shapes. In one variation the paper is made to move down, then up, down again and finally up, by means of three idler rollers, thus shortening the length of cooling chamber 51.

It is to be noted that the present invention is not to be confused with other devices or arrangements of the prior art herein explained. This technique of continuous antidromic electrophoresis as used with the instruments shown in the drawings and described in the example has the advantage of the ability to perform separations not attainable before by electrophoresis. It has the further advantages of compactness, lower voltages, approximately linear rates of migration, and ease of construction and operation. Furthermore, although the present invention has been described in conjunction with preferred embodiments, it is to be understood that modifications and variations may be resorted to without departing from the spirit and scope of the invention, as those skilled in the art will readily understand. Such modifications and variations are considered to be within the purview and scope of the invention and appended claims.

I claim:

1. An instrument for chemical identification, for the separation of substances whose electrical ions have slightly different rates of electrophoretic migration, the separations being reproducible and at constant relative distances from each other, comprising in combination; an elongated frame to define the limits of an electrically conductive absorbent carrier tape; a feed reel at one end of said frame to contain and feed out a continuous even supply of said carrier tape; guide rollers towards said one end of the frame to maintain saidcarrier tape suspended along the length of said frame; a pair of buffer solution containers towards opposing ends of said frame; electrical terminals in said frame, lead lines from said terminals to electrodes which enter said containers to provide positive and negative electricity to a buffer solution in said containers; impurity retaining means adjacent said electrodes in said containers preventing impurities formed by the electrical activity from being carried along in a buffer solution in said containers; wick roller contacts for evenly moistening the buffer solution onto the carrier tape and conducting electricity onto and off said tape, said contacts being housed in said containers at opposing ends of said frame; a takeup reel at the other end of said frame to take up and retain said carrier tape; rollers for moving said carrier tape towards said other end; slow constant speed moving motor means and gearing coupling said motor means and rollers for continuously and constantly moving said carrier tape at a uniform slow speed somewhere of about the speed of electrophoretic migration of the substances to be separated; a cooling chamber intermediate said wick roller contacts, to contain an inert heat transfer fluid; and, a cooling coil in said cooling chamber to circulate a cooling fluid maintained at a constant temperature.

2. An apparatus for the repeated separations of substances whose electrical ions have slightly different rates of electrophoretic migration, the separations being reproduced and at constant relative distances from each other, comprising in combination; an elongated frame to support and define the limits of an absorbent elongated supporting medium for absorbing an electrically conductive buifer solution, said frame also defining a bufier solution container towards each end thereof to retain a buffer solution therein; feed reel means at one end of said frame to contain and reel out a continuous even supply of a long flexible supporting medium; guide means along said frame to guide said supporting medium along the length thereof; electrical terminals in said frame, lead lines from said terminals to electrodes which enter said containers to provide positive and negative electricity to a buffer solution in said containers; contact means separated by a fixed distance for engaging said supporting medium towards each end of said frame to supply electric current to said supporting medium by evenly moistening said supporting medium with said bufier solution; reel take-up means at the other end of said frame to take up and retain said supporting medium; constant speed motor means including a driven means for continuously moving said carrier medium at a uniform slow speed of about the speed of electrophoretic migration of the chemical substances to be separated; and, cooling means intermediate said contact means in the path of travel of said supporting medium, for maintaining said supporting medium at constant temperature.

3. An apparatus as claimed in claim 2 including a carrier tape extending across said frame between said feed reel and reel tape-up means.

4. An apparatus as claimed in claim 3 including a gel coated supporting medium supported by said frame between said feed reel means and said reel tape-up means.

5. An apparatus as claimed in claim 3, said contact means being wick roller contacts.

6. An instrument as claimed in claim 3 having an elongated cooling chamber intermediate said contact means for engaging said supporting medium, said elongated chamber being disposed at the center of said frame, substantially at right angles to said frame; guide rollers on both sides of said chamber changing the direction of travel of said tape in and out of said chamber by about 90 degrees; guide means in said chamber reversing the direction of travel of said tape; and cooling conducting elements in said chamber, said chamber being designed to contain chemically non-reactive electrically coolable cooling medium to cool said tapes as it travels through said chamber and maintain it at a constant temperature.

References Cited in the file of this patent UNITED STATES PATENTS Re. 24,752 Ressler Dec. 15, 1959 2,566,308 Brewer Sept. 4, 1951 2,762,770 Widmer et a1 Sept. 11, 1956 2,768,948 McDonald Oct. 30, 1956 2,843,540 Ressler July 15, 1958 2,878,178 Bier Mar. 17, 1959 2,879,217 Durrum et al Mar. 24, 1959 2,884,367 Karler et al. Apr. 28, 1959 FOREIGN PATENTS 791,570 Great Britain a Mar. 5, 1958 OTHER REFERENCES Bonnin: Comptes Rendus, vol. 244, The Academy of Sciences, Paris, 1957, pages 2708-10 relied upon.

Chemlo, M.: Chromatographic Reviews, vol. I, Elsevier Pub. Co., New York, January 1959, pages 254-56. I

Bonnin et al.: Comptes Rendus, vol. 241, The Academy of Sciences, Paris, 1955, pages -42 relied upon.

Chemical Products, July 1954, pages 260-266.

Bermes et al.: Chromatographic Methods, vol. 2, No. 1, March 1957, pages 1-5.

Fox: Apparatus for Chromatographic and Electrophoretic Analysis, Chemical Products, July 1954, pages 260-266. 

1. AN INSTRUMENT FOR CHEMICAL IDENTIFICATION, FOR THE SEPARATIN OF SUBSTANCES WHOSE ELECTRICAL IONS HAVE SLIGHTLY DIFFERENT RATES OF ELECTROPHORETIC MIGRATION, THE SEPARATIONS BEING REPRODUCIBLE AND AT CONSTANT RELATIVE DISTANCE FROM EACH OTHER, COMPRISING IN COMBINATION; AN ELONGATED FRAME TO DEFINE THE LIMITS OF AN ELECTRICALLY CONDUCTIVE ABSORBENT CARRIER TAPE; A FEED REEL AT ONE END OF SAID FRAME TO CONTAIN AND FEED OUT A CONTINUOUS EVEN SUPPLY OF SAID CARRIER TAPE; GUIDE ROLLERS TOWARDS SAID ONE END OF THE FRAME TO MAINTAIN SAID CARRIER TAPE SUSPENDED ALONG THE LENGTH OF SAID FRAME; A PAIR OF BUFFER SOLUTION CONTAINERS TOWARDS OPPOSING ENDS OF SAID FRAME; ELECTRICAL TERMINALS IN SAID FRAME, LEAD LINES FROM SAID TERMINALS TO ELECTRODES WHICH ENTER SAID CONTAINERS TO PROVIDE POSITIVE AND NEGATIVE ELECTRICITY TO A BUFFER SOLUTION IN SAID CONTAINERS; IMPURITY RETAINING MEANS ADJACENT SAID ELECTRODES IN SAID CONTAINERS PREVENTING IMPURITIES FORMED BY THE ELECTRICAL ACTIVITY FROM BEING CARRIED ALONG IN A BUFFER SOLUTION IN SAID CONTAINERS; WICK ROLLER CONTACTS FOR EVENLY MOISTENING THE BUFFER SOLUTION ONTO THE CARRIER TAPE AND CONDUCTING ELECTRICITY ONTO AND OFF SAID TAPE, SAID CONTACTS BEING HOUSED IN SAID CONTAINERS AT OPPOSING ENDS OF SAID FRAME; A TAKEUP REEL AT THE OTHER END OF SAID FRAME TO TAKE UP AND RETAIN SAID CARRIER TAPE; ROLLERS FOR MOVING SAID CARRIER 